Apoptosis is crucial for normal development and homeostasis in metazoans (Fuchs Y and Steller H, Cell 147, 742-758 (2011); incorporated by reference herein). Mammals and lower vertebrates have evolved a unique signaling mechanism, termed apoptosis that, under certain circumstances, programs individual cells to die (Elmore S, Toxicol Pathol 35, 495-516 (2007); incorporated by reference herein). Alternatively, induction of apoptosis is essential for the elimination of oncogenically transformed cells. Multiple cellular pathways triggering apoptosis are described. Over the years, the two main apoptotic pathways, the extrinsic and intrinsic pathways, have been studied in a great detail. The extrinsic pathway involves the interaction of ligands, including TNF, FasL, and TRAIL, with their respective receptors and the consequential activation of the downstream caspases and Bcl-2 family members (Fulda S and Debatin K M, Oncogene 25, 4798-4811 (2006) and Tait S W and Green D R, Nat Rev Mol Cell Biol 11, 621-632 (2010); both of which are incorporated by reference herein). The intrinsic pathway is triggered by internal signals (e.g., DNA damage) that are produced following cellular stress and involves the mitochondria. Initiation of either pathway results in the activation of chain-like caspases followed by proteolysis of cellular proteins and the degradation of chromosomal DNA.
TRAIL is able to trigger apoptosis in a variety of tumor cells but not in normal cells (Ashkenazi A et al, J Clin Invest 104, 155-162 (1999); Walczak H et al, Nat Med 5, 157-163 (1999); Pittim R M et al, J Biol Chem 271, 12687-12690 (1996); Wang S, Oncogene 27, 6207-6215 (2008); and Ashkenazi A, Cytokine Growth Factor Rev 19, 325-331 (2008); all of which are incorporated by reference herein). TRAIL is a type II membrane protein and, similar to TNF-α, TRAIL can be shed from the cell surface membrane to produce a soluble, biologically active form. Expression of TRAIL transcripts has been detected in many human tissues, mostly in the spleen, lung, and prostate (Wiley S R et al, Immunity 3, 673-682 (1995) and Ashkenazi A, Nat Rev Cancer 2, 420-430 (2002); both of which are incorporated by reference herein). TRAIL forms homotrimers with a stoichiometric zinc atom bound by the cysteine residue of each molecule in the trimeric ligand. Zn stabilizes the TRAIL homotrimer and is essential for its biological activity (Hymowitz S G et al, Mol Cell 4, 563-571 (1999) and Bodmer J L et al, J Biol Chem 275, 20632-20637 (2000); both of which are incorporated by reference herein).
TRAIL induces apoptosis by utilizing components of both the extrinsic and intrinsic cellular pathways (Song J H et al, Brain Pathol 13, 539-553 (2003) and Falschlehner C et al, Intl Biochem Cell Biol 39, 1462-1475 (2007); both of which are incorporated by reference herein). In the extrinsic pathway, apoptosis is initiated by the interaction of TRAIL with its respective death receptors, DR4 and DR5. These interactions lead to the receptor trimerization, to the clustering of the receptor's intracellular death domains (DD), and to the formation of the death-inducing signaling complex (DISC). DISC formation leads to the recruitment of an adaptor molecule, FADD, with the subsequent binding and activation of apical caspase-8 and apical caspase-10. Activated caspase-8 and -10 then cleave and activate the ‘executioner’ caspases-3 and other downstream caspases, followed by the cleavage of the death substrates and, eventually, cell death.
The TRAIL-induced intrinsic pathway involves the cleavage of the proapoptotic Bcl-2 family member Bid by active caspase-8. Truncated Bid is then translocated to the mitochondria where it promotes the release of cytochrome c and SMAC/DIABLO into the cytosol via interactions with the proapoptotic proteins Bax and Bak (Wei M C et al, Cancer Res 63, 1712-1721 (2003); incorporated by reference herein). By binding to the adaptor protein APAF-1, cytochrome c induces the formation of ‘apoptosome’, a structure that activates caspase-9. Proteolytically active caspase-9 in turn causes activation of the so called ‘executioner’ proteases (caspases-3, -6, and -7) in the presence of dATP, which leads to the cleavage of the death substrates. Antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL block cytochrome c release and, therefore, are negative regulators of the intrinsic apoptotic pathway (Cheng E H et al, Mol Cell 8, 705-711 (2001); incorporated by reference herein). TRAIL, by employing both the extrinsic and intrinsic apoptosis signaling pathways, amplifies the apoptotic signal initiated through its binding to the death receptors. The existence of two signaling apoptotic pathways mediated by TRAIL reveals the existence of two different cell types (Lemke J et al, Cell Death Differ 21, 1350-1364 (2014); incorporated by reference herein). In one cell type (type I), the apoptotic pathway is independent of the mitochondria and depends on the caspase-8 activation followed by the activation of effector caspases such as caspase-3. In the other cell type (type II), apoptosis is dependent on the amplification of the apoptotic signal via the mitochondrial (intrinsic) pathway. Overexpression of the anti-apoptotic Bcl-2 protein does not affect apoptosis in type I cells, but blocks apoptosis in type II cells.